Throughout and within this application are referenced publications, the full bibliographic citations for which are found within the text of the application or at the end of the specification, immediately preceding the claims. The disclosures of these publications, as well as published patent specifications, books and issued patents are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.
It is well known that pharmaceutical research leading to the identification of a new drug generally involves the screening of very large numbers of candidate substances, both before and after, a lead component has been found. This is one factor which makes pharmaceutical research very expensive and time-consuming, so that a method for assisting in the screening process can have considerable commercial importance and utility.
Many pharmaceuticals exert their therapeutic effect by interacting with DNA in the cell. Some pharmaceuticals are targeted to the correction of genetic abnormalities, the accumulation of which leads to a diseased state. For example, mutations in genes commonly occurs during cancer progression and can greatly elevate the frequencies of base alterations or large scale chromosome rearrangements. For example, defects in cell cycle control pathways involving the p53 tumor suppressor gene create a permissive environment in which cells with aneuploidy, chromosome translocations and gene amplification arise at high frequency in response to stresses created by antimetabolites or oncogene overexpression (Livingstone, et al. (1992); Yin, et al. (1992) and Denko, et al. (1994)).
The types of aberrant chromosomal structures generated in cells with defective repair and cell cycle control functions are likely to be constrained by nuclear structure. For example, chromosomes with very long arms tend to generate nuclear projections variously referred to as “blebs” or “buds” occur (Ruddle (1962); Lo and Fraccaro (1974); Toledo, et al. (1992) and Pedeutour, et al. (1994)). A recent study in peas demonstrated that excessive DNA within a single chromosome arm generated a nuclear projection which was cut when the cell division plate formed after telophase (Schubert and Oud (1997)). Sequences enclosed in such projections are often detected in micronuclei, suggesting that projections can be precursors of micronuclei (Toledo, et al. (1992) and Pedeutour, et al. (1994)), and that the chromosomal sequences they contain can be lost from the nucleus. These data indicate that a maximum allowable size exists for each chromosome arm within the nuclei of specific cell types.
Circular, autonomously replicating DNA fragments such as double minute chromosomes (DMs) also frequently generated in cancer cells (Barker (1982); Cowell (1982) and Benner, et al. (1991)). These structures encode proteins that provide survival advantages in vivo, or resistance to a variety of chemotherapeutic agents in vitro (see Wahl (1989); Brison (1993); Von Hoff, et al. (1992); Shimizu, et al. (1994) and Eckhardt, et al. (1994)). DMs replicate using cellular replication origins (Carroll, et al. (1993)), but lacking centromeres, they do not segregate by the same mechanism employed by chromosomes. Consequently, DMs are lost spontaneously in the absence of selection. Drugs such as hydroxyurea significantly increase the loss rate of DMs in human and rodent cell lines (Snapka and Varshavsky (1983); Von Hoff, et al. (1991); Von Hoff, et al. (1992); Eckhardt, et al. (1994) and Canute, et al. (1996)). DM elimination results in increased drug sensitivity, reduced tumorigenicity, or differentiation, depending on the proteins expressed by DM-encoded genes (Snapka and Varshavsky (1983); Snapka (1992); Von Hoff, et al. (1992); Eckhardt, et al. (1994) and Shimizu, et al. (1994)). For this reason, identifying the mechanisms by which DMs are eliminated could enable the development of new or more selective chemotherapeutic strategies since DMs are uniquely found in cancer cells, and chromosome loss should not be induced by such treatments.
Like abnormally long chromosome arms, DMs have also been reported to be preferentially incorporated within micronuclei that are removed from the cell (Von Hoff, et al. (1992) and Shimizu, et al. (1996)). It is clear that small size alone does not guarantee selective enclosure of DNA fragments within micronuclei since a centric minichromosome the size of a typical DM is effectively excluded from micronuclei (Shimizu, et al. (1996)). This observation is consistent with the classical mechanism of micronucleus formation which involves the enclosure of lagging acentric chromosome fragments as nuclear membranes reform at the end of mitosis (Heddle and Carrano (1977) and Heddle, et al. (1983)). Thus, one would expect post-mitotic enclosure of DMs within micronuclei since they typically lack functional centromeres (Levan, et al. (1976)). However, DMs appear to associate with chromosomes or nucleoli, which may enable most of them to evade such a post-mitotic mechanism. The ability of DMs to “hitch-hike” by association with mitotic chromosomes or nucleoli provides one explanation why few micronuclei were detected at the midbody in a cell line containing numerous DMs (Levan and Levan (1978)), and their surprisingly efficient partitioning to daughter cells in some cell lines (Levan and Levan (1978) and Hamkalo, et al. (1985)). However, the interphase behavior of normal chromosomes and DMs may differ because DMs lack the centromeres and/or telomeres which position chromosomes in restricted territories and produce a choreographed set of chromosome movements during S-phase (De and Mintz (1986) and Cremer et al. (1993)). It has not been determined whether acentric DM DNA occupies positions different from chromosomes in interphase, and whether this could enable their removal from the nucleus by a budding process like that observed for abnormally long chromosomes (Ruddle (1962); Jackson and Clement (1974); Lo and Fraccaro (1974); Miele, et al. (1989) and Toledo, et al. (1992)).